Mercuric ion reductase is a homodimeric enzyme with two active sites for catalysis. The active sites of the enzyme, although nominally identical, exhibit spectroscopic differences and different reactivities when the enzyme is complexec with substrate and/or product ligands. In the absence of ligands the active site environments appear to be identical. The observation of asymmetry may play an essential role in the catalytic mechanism of the enzyme. The two major goals of this project: (1) elucidating the structural pathways through which the active sites of the enzyme communicate upon ligand binding, and (2) elucidating the role of intersite communication in the catalytic mechanism, i.e., is it essential that the dimers be asymmetric during catalysis or can the sites operate independent of one another. As one approach to investigating how asymmetry is induced upon ligand binding, we are synthesizing modified flavin cofactors with appropriate redox properties and unique spectroscopie properties that will be sensitive to changes in the local environment of the active sites when ligands bind. Using these modified flavins, we hope to identify which ligands or parts of the ligands are necessary and sufficient to induce asymmetry in the active sites. This information can then be combined with evaluation of crystal structure data to identify potential structural pathways for communication between the active sites. Our current need for mass spectrometry in this project is for the characterization of our synthetic flavin cofactors.